Project Summary/Abstract Section The Corynebacterineae suborder encompasses many infectious agents, including deadly human pathogens, such as Mycobacterium tuberculosis (M. tb). Many antibiotics used to treat M. tb block steps in cell envelope assembly, underscoring the key role of this protective barrier. The Corynebacterineae cell envelope is unique in that it is constructed from saccharide and lipid building blocks that differ dramatically even from those used by other bacteria. One of the central features of the Corynebacterineae cell envelope is the mycolyl arabinogalactan (mAG), which is a glycoconjugate assembled from galactofuranose (Galf), arabinofuranose (Araf), and mycolic acids. Most enzymes involved in mAG biosynthesis have been identified but when, where, and how they function in live cells to construct and remodel the cell envelope is not known. To elucidate these changes and their functional roles, the proposed aims focus on developing new tools to probe, perturb, and exploit changes in the Corynebacterineae cell envelope. The focus of Aim 1 is on generating a small molecule probe of the galactan?the only major cell wall constituent for which no effective probes exist. The target enzyme is UDP- galactopyranose mutase (UGM), which catalyzes the formation of the key building block required for biosynthesis of the essential galactan. The small molecule probe that results can be used to evaluate the consequences of blocking galactan assembly to understand the role of galactan in the stability and integrity of the cell envelope in Corynebacterineae and to probe the role of UGM in other organisms. Aim 3 focuses on the mycolyltransferases, which construct the critical mycolic acid components of the cell envelope. The localization of these enzymes and the timing of their activity in cell division is not known. To visualize mycolyltransferase activity in live cells, we propose to devise fluorogenic probes that can pinpoint the sites of Ag85 activity and reveal the interplay between mycolyltransferase activity and cell envelope assembly. We anticipate that the probes described in Aim 3 can be used to develop assays for finding inhibitors of cell division and for monitoring and detecting mycobacteria in real time. We expect that our pursuit of the three aims shall uncover vulnerabilities in the defenses of mycobacteria and corynebacteria that will lead to new antibiotic strategies. Significance: The overall objective of this application is to develop new chemical probes to understand how the Corynebacterineae build and maintain their cell wall to survive. We anticipate that this knowledge will lead ultimately to the identification of new strategies to treat infectious disease.